PROJECT SUMMARY Spine fusion and pectus repair are among the most painful musculoskeletal surgeries adolescents undergo, with a high incidence of chronic postsurgical pain (CPSP). CPSP is detrimental to recovery and increases the risk for disability as well as prescription opioid abuse. Understanding CPSP risk is important for development of effective preventive and therapeutic strategies. Although genetic, psychosocial and environmental factors explain some of the variance in CPSP risk, there remain critical gaps in CPSP risk prediction and understanding of longitudinal gene-environmental influences on long-term pain responses after surgery. This leads us to our global hypothesis that epigenetic processes will influence the development and evolution of CPSP. DNA methylation (DNAm) is a key epigenetic mechanism known to influence transcription and transition of acute to chronic pain. Preliminary data: Our pilot epigenome-wide association study (EWAS) in spine surgical subjects showed that CPSP is associated with differential DNAm in 39 genes which enrich GABA, Dopamine-DARPP32 Feedback in cAMP and immune signaling pathways. Our previous findings that DNAm of Mu opioid receptor gene promoter, perioperative pain and opioid exposures predict CPSP, lead us to believe that DNAm mediates the association of perioperative stressors with long-term pain phenotypes. Increased cytokine levels and DNAm-expression correlations in CPSP (pilot data) suggest differential epigenetic regulation influencing immune cell-specific gene expression in CPSP. Scientific objectives are to determine blood DNAm biomarkers for CPSP and evaluate temporal origins of CPSP-associated epigenetic variation in relation to pain-opioid exposures and inflammatory responses. The premise for the potential utility of blood based DNAm as CPSP biomarkers is supported by cross tissue comparison studies of brain-blood DNAm correlations and blood DNAm signatures reported for several neural phenotypes. Aim 1: Determine and validate differentially methylated regions predictive of CPSP. The working hypothesis is that CPSP will be significantly associated with DNAm markers in preoperative blood samples after adjusting for demographic, psychosocial and perioperative factors. We will conduct a prospective, multisite EWAS for discovery-validation (N=1165 opioid-nave children undergoing spine fusion) and replication of candidate markers (N=300 children undergoing pectus repair). Since cross-sectional epigenetic studies are potentially affected by reverse causation bias and genetic variation confounders, we propose novel longitudinal studies to evaluate perioperative stress related DNAm changes. Aim 2: Determine longitudinal changes in DNAm profiles associated with pain and opioid exposures and explore their role in mediating associations with CPSP. Aim 3: Characterize cytokine profiles, immune cell subsets and epigenetic regulation of immune cell specific gene expression in CPSP. It is anticipated that this study will identify novel epigenetic biomarkers for CPSP, provide novel insights into longitudinal epigenetic mechanisms in the evolution of CPSP, and uncover promising therapeutic targets for CPSP, with extended implications for other chronic pain conditions.